The long range goal of this project is to provide a detailed biochemical understanding of endogenous liver microsomal isozymes of cytochrome P-450 which catalyze key biosynthetic reactions. The initial focus of this project is on rat liver cholesterol 7 Alpha-hydroxylase, which catalyzes the first and rate-limiting step in the conversion of cholesterol to bile acids. This metabolic conversion accounts for some 50 to 80% of the total turnover of exchangeable cholesterol pools in both rats and in mice. A reduction of body bile acid pools, in some cases reflecting decreased liver cholesterol 7 Alpha-hydroxylase activity, is believed to be a primary cause of the supersaturation of bile with cholesterol, leading to gallstone formation. The major objectives of this project are (1) to purify cholesterol 7 ALpha-hydroxylase to homogeneity from rat liver, and then study its biochemistry and interactions with other microsomal electron transfer proteins in a reconstituted monooxygenase system, (2) to evaluate the biochemical relationships between P-450 isozymes, such as cholesterol 7 Alpha-hydroxylase, that function physiologically in oxidative metabolism of endogenous substrates, and those that are inducible by xenobiotics and appear to function primarily in foreign compound metabolism and (3) to examine various regulatory mechanisms proposed for cholesterol 7 Alpha-hydroxylase and then assess their roles in modulating this rate-limiting enzyme of cholesterol degradation in vivo. Studies of the biochemistry and regulation of cholesterol 7 Alpha-hydroxylase may shed light on metabolic defects leading to cholesterol stone formation and, in addition, may suggest medically useful approaches to gallstone disease.